Transcript Lecture5_6 - Welcome to people.pharmacy.purdue.edu!

• Office Hours
• Color slides
Questions From Class
Is IgM only pentameric?
IgM is made as a monomer in the cell and then assembled in
the cell into a pentamer, with the help of the J chain. After
assembly, it is secreted from the cell as a pentamer.
Therefore, the secreted form of IgM is only present as a
pentamer
Why does a T helper cell (CD4 TH1 have to activate a
macrophage after the same macrophage has already
phagocytosed a pathogen?
Macrophages have two main effector functions (see Fig
1.14): 1) phagocytosis and 2) effector signaling by secretion
of cytokines. TH1 cells will themselves secrete cytokines to
increase the overall effector activity of the macrophage
including the secretion of cytokines from the same
macrophage (Fig 1.27, 3.11 and 8.15).
Questions From Class (continued)
What does soma mean with respect to somatic
recombination?
Soma refers to all the cells of the body (soma is latin for
body) except germ cells. Germline configuration refers to the
arrangement of genes in all of the somatic cells and germ
cells that do not undergo somatic recombination. In cells that
have the unique capability to undergo somatic recombination,
such as B and T cells, germline configuration refers to gene
arrangement before those cells have undergone gene
rearrangement (by the process of somatic recombination)
A) Can the two types of light chain( and ) be made at the
same time in the same B cell? B) How does that affect Ab
diversity?
A) No. One B cell only expresses one type of light chain. B)
Please see the following slide for the correct calculation.
Random Recombination of
Gene segments is one
factor contributing to
diversity
(200* + 120*)
X
10,530 = 3,369,600 Ig molecules
* Only one light chain loci gives rise to one functional polypeptide!
Generation of Ig diversity in B cells
Unique organization
- Only B cells can express Ig protein
- Gene segments
  - Light chain
, , , ,  - Heavy Chain
present on three chromosomes
The Gene Rearrangement
Concept
• Germline configuration
• Gene segments need to be
reassembled for expression
• Sequentially arrayed
• Occurs in the B-cells precursors
in the bone marrow (soma)
• A source of diversity BEFORE
exposure to antigen
V-variable, J-joining, D-diversity gene segments; L-leader sequences
Light Chain
Variable - V, J
Constant - C
Heavy Chain
Variable - V, D, J
Constant - C
Gene rearrangements during B-cell development
V-variable, J-joining, D-diversity gene segments; L-leader sequences
Figure
2-14
 - 30 V & 4 pairs J & C (light chain) chs22
 – 40 V & 5 J & 1 C (50% have 2x V) (light chain) chs2
H – 65 V & 27 D & 6 J
chs14
J-joining
Figure 2-15 part 1 of 2
CDR1 and CDR2
CDR3
D-diversity
Figure 2-15 part 2 of 2
CDR1 and CDR2
CDR3
Random Recombination of
Gene segments is one
factor contributing to
diversity
(200* + 120*)
X
10,530 = 3,369,600 Ig molecules
* Only one light chain loci gives rise to one functional polypeptide!
Mechanism of Recombination
• Recombination signal sequences (RSSs) direct recombination
- V and J (L chain)
- V D J (H chain)
• RSS types consist of:
- nonamer (9 base pairs)
- heptamer (7 base pairs)
- Spacer
- Two types: [7-23-9] and [7-12-9]
• RSS features:
- recognition sites for recombination enzymes
- recombination occurs in the correct order
(12/23 rule)
Mechanism of Somatic Recombination
• V(D)J recombinase: all the protein components that mediate the
recombination steps
• RAG complex: Recombination Activating Genes (RAG-1 and RAG2) encode RAG proteins only made in lymphocytes, plus other
proteins
• Recombination only occurs through two different RSS bound by two
RAG complexes (12/23 rule)
• DNA cleavage occurs to form a single stranded hairpin and a break at
the heptamer sequences
• Enzymes that cut and repair the break introduce Junctional Diversity
Recombination
+
Junctional
Diversity
Figure 2-19
Junctional Diversity
Figure 2-18 part 2 of 3
• Nucleotides introduced at recombination break in the coding joint
corresponding to CDR3 of light and heavy chains
- V and J of the light chain
- (D and J) or (V and DJ) of the heavy chain
• P nucleotides generate short palindromic sequences
• N nucleotides are added randomly - these are not encoded
• Junctional Diversity contribute 3 x107 to overall diversity!
Generation of BCR (IgD and IgM)
• Rearrangement of VDJ of the heavy chain brings the gene’s promoter
closer to C and C
• Both IgD and IgM are expressed simultaneously on the the surface of
the B cell as BCR - ONLY isotypes to do this
• Alternative splicing of the primary transcript RNA generates IgD and
IgM
• Naïve B cells are early stage B cells that have yet to see antigen and
produce IgD and IgM
Alternative Splicing of Primary Transcript to
generate IgM or IgD
Figure 2-21
Summary Biosynthesis of IgM in B cells
Mature B cell
Figure 2-23
•
Long cytoplasmic tails
interact with intracellular
signaling proteins
•
Disulfide-linked
•
Transmembrane proteins invariant
•
Dual-function
1) help the assembled Ig
reach the cell surface from
the ER
2) signal the B cell to divide
and differentiate
Principle of Single Antigen Specificity
• Each B cell contains two copies of the Ig locus (Maternal and
Paternal copies)
• Only one is allowed to successfully rearrange - Allelic Exclusion
• All Igs on the surface of a single B cell have identical specificity
and differ only in their constant region
• Result: B cell monospecificity means that a response to a pathogen
can be very specific
DNA hybridization of Ig genes can diagnose B-cell leukemias
Generation of B cell diversity in Ig’s before Antigen
Encounter
1. Random combination of V and J (L chain) and V, D, J (H chain)
regions
2. Junctional diversity caused by the addition of P and N nucleotides
3. Combinatorial association of Light and Heavy chains
(each functional light chain is found associated with a different
functional heavy chain and vice versa)
Developmental stages of B cells
1. Development
before antigen
Immature B cell
2. Development
after antigen
Mature naive B cell
(expressing BCR IgM and IgD)
plasma cell
(expressing BCR and
secretes Antibodies)
Processes occurring after B cells encounter antigen
•
Processing of BCR versus Antibody
1. Plasma cells switch to secreted Ab
2. Difference occurs in the c-terminus of the heavy chain
3. Primary transcript RNA is alternatively processed to
yield transmembrane or secreted Ig’s
•
Somatic Hypermutation
1. Point mutations introduced to V regions
2. 106 times higher mutation rate
3. Usually targets the CDR
•
Affinity maturation - mutant Ig molecules with higher affinity
are more likely to bind antigen and their B cells are
preferentially selected
•
Isotype switching
RNA processing to generate BCR or Antibody
MC - membrane coding
SC - secretion coding
Somatic Hypermutation
(random introduction of point mutations)
Mutations occur throughout the V domain - especially CDR
Occurs on both gene copies - but only one expresses protein
AID - Activation induced Cytidine Deaminase
UNG - Uracil-DNA glycosylase
Process of Affinity Maturation
IgM
Hypermutation
IgG
IgM
Isotype switching
1. IgM is the first Ab that is secreted in the IR
2. IgM is pentameric and each H chain can bind complement proteins
3. Isotypes with better effector functions are produced by activated B
cells
4. Rearrangement of DNA using SWITCH regions
- all C genes preceded by switch sequence (except 
- start from the  gene and any other C gene (plus sequential)
5. Regulated by cytokines secreted by T cells
Switch regions
Mu to any other isotype
Sequential switching
AID is important
Immunoglobulin classes
Figure 2-31 part 2 of 2
1. C regions determine the class of antibody and their effector
function
2. Divided into Subclasses based on relative abundance in serum
3. Each class has multiple functions
1. IgM and IgG can bind complement
2. IgG crosses placenta
3. Receptors for constant regions (Fc Receptors)
- IgG (FcG receptors): mac, neutrophils, eosinophils,
NK cells, others
- IgE (FcE receptors): mast cells, basophils, others
Initial Immune Response mediated by IgM
IgM
(plasma cells in
lymph nodes,
spleen, and bone
marrow and
circulate in
blood/lymph)
Low affinity
binding to antigen
via multiple
binding sites
Two binding sites
sufficient for
strong binding
Hypermutation
and affinity
maturation
Exposure of
constant region
Activate
complement
Phagocytose
Isotype switching to IgG
Kill
directly
IgG
(lymph nodes,
spleen, and
bone marrow)
Circulates in
blood and lymph
(most abundant
Ab in internal
fluids)
Extravasation,
Higher affinity
binding to
antigen
Recruit
phagocytes
Multiple
effector
functions
Neutralize
antigens
Activate
complement
Monomeric IgA
Plasma cells in
lymph nodes,
spleen, bone
marrow
Dimeric IgA
lymphoid tissue
associated with
mucosal surfaces
Secreted into
Blood
Secreted into Gut
lumen & body
secretions
Effector functions
1. Mainly
neutralization
2. Minor
opsonization
and activation
of complement
IgE
Plasma cells in
lymph nodes or
germinal centers
Bind strongly to
Mast cells
Inflammation
- Expulsion of large pathogens
- Allergies
Cross-linking of
receptor bound
Ab releases
histamine and
other activators
Figure 2-32
Summary: Generation of B-cell diversity
• Diversity before Antigen exposure (Antigen Independent)
- Random Recombination
- Junctional Diversity
- Combinatorial association
• Diversity after Antigen exposure (Antigen Dependent)
- Switch to secreted Ab
- Somatic Hypermutation
- Affinity Maturation
- Isotype Switching
• Immunoglobulin Classes
- Properties
- Effector functions